Humanized C5 animals

ABSTRACT

Non-human animals comprising a human or humanized C3 and/or C5 nucleic acid sequence are provided as well as methods for using the same to identify compounds capable of modulating the complement system. Non-human animals that comprise a replacement of the endogenous C5 gene and/or C3 gene with a human or humanized C5 gene and/or C3 gene, and methods for making and using the non-human animals, are described. Non-human animals comprising a human or humanized C5 gene under control of non-human C5 regulatory elements is also provided, including non-human animals that have a replacement of non-human C5-encoding sequence with human C5-encoding sequence at an endogenous non-human C5 locus. Non-human animals comprising a human or humanized C3 gene under control of non-human C3 regulatory elements is also provided, including non-human animals that have a replacement of non-human C3 protein-encoding sequence with human or humanized C3 protein-encoding sequence at an endogenous non-human C3 locus. Non-human animals comprising human or humanized C3 and/or C5 sequences, wherein the non-human animals are rodents, e.g., mice or rats, are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Non Provisional patentapplication Ser. No. 14/703,818, filed May 4, 2015, which claimspriority to U.S. Provisional Patent Application No. 61/988,581, filedMay 5, 2014, and U.S. Provisional Patent Application No. 62/067,836,filed Oct. 23, 2014, the disclosures of each of which are incorporatedby reference herein in their entirety.

FIELD OF INVENTION

Disclosed herein are non-human animals comprising nucleic acid sequencesencoding a C3 protein and/or a C5 protein that comprise a human sequenceas well as transgenic non-human animals comprising a C3 and/or a C5 genethat is human in whole or in part. Also disclosed herein are non-humananimals that express human or humanized C3 and/or C5 proteins andmethods for using non-human animals comprising human or humanized C3and/or C5 nucleic acid sequences.

BACKGROUND

Complement proteins C5 and C3 are therapeutic targets for treatment of avariety of human diseases, disorders and conditions that are associatedwith complement activation, for example, ocular inflammatory and retinaldegenerative diseases. The evaluation of pharmacokinetics (PK) andpharmacodynamics (PD) of therapeutic molecules that specifically targethuman C5 or human C3 proteins are routinely performed in non-humananimals, e.g., rodents, e.g., mice or rats. However, the PD of suchtherapeutic molecules cannot properly be determined in certain non-humananimals because these therapeutic molecules do not target the endogenousC5 or C3 proteins.

Moreover, the evaluation of therapeutic efficacy of human-specific C5 orC3 protein antagonists various non-human animal models of diseasesassociated with an activated complement system is problematic innon-human animals in which such species-specific antagonists do notinteract with the endogenous C5 or C3 proteins.

Accordingly, there is a need for non-human animals, e.g., rodents, e.g.,murine animals, e.g., mice or rats, in which the C5 and/or C3 genes ofthe non-human animal are humanized in whole or in part or replaced(e.g., at the endogenous non-human loci) with human C5 and/or C3 genescomprising sequences encoding human or humanized C5 and/or C3 proteins,respectively. There is a need for such humanized non-human animals thatexpress human or humanized C5 and/or C3 proteins in serum atconcentrations similar to that of C5 and/or C3 proteins, respectively,present in serum of an age-matched non-human animal, that expressesfunctional C5 and/or C3 proteins, but does not comprise the human orhumanized C5 and/or C3 genes, respectively.

Throughout this specification, various patents, patent applications andother types of publications (e.g., journal articles, electronic databaseentries, etc.) are referenced. The disclosure of all patents, patentapplications, and other publications cited herein are herebyincorporated by reference in their entirety for all purposes.

SUMMARY

Non-human animals comprising nucleic acid sequences encoding a C3 and/ora C5 that comprise a human sequence are provided.

Transgenic non-human animals comprising a C3 and/or a C5 gene that ishuman in whole or in part are provided.

Non-human animals that express human or humanized C3 and/or C5 proteinsare provided.

Non-human animals having a replacement (in whole or in part) ofendogenous non-human animal C5 and/or C3 genes are provided.

Non-human animals comprising a C5 and/or C3 humanization (in whole or inpart) at an endogenous non-human C5 and/or C3 locus are provided.

Non-human animals are provided that have human or humanized C5 and/or C3genes, wherein the non-human animals do not express endogenous C5 and/orC3 protein, and express human or humanized C5 and/or C3 protein, inserum at concentrations similar to that of C5 and/or C3 proteins,respectively, present in serum of an age-matched non-human animal thatexpresses functional endogenous C5 and/or C3 proteins, but does notcomprise the replacement.

In one aspect, non-human animals comprising a human or humanized C3and/or C5 nucleic acid sequence are provided.

In one aspect, genetically modified non-human animals are provided thatcomprise a replacement at an endogenous C5 and/or C3 locus of a geneencoding an endogenous C5 and/or C3 gene encoding a human or humanizedC5 and/or C3 protein. Rodents, e.g., mice or rats, are provided thatcomprise a replacement of an endogenous C5 gene, at an endogenous rodentC5 locus, with a human C5 gene, and/or comprise a replacement of anendogenous C3 gene, at an endogenous rodent C3 locus, with a human C3gene. In one embodiment, the rodent is a mouse. In one embodiment, therodent is a rat.

In one aspect, genetically modified rodents, e.g., mice or rats, areprovided comprising a replacement at an endogenous rodent C5 locus of arodent gene encoding C5 protein with a human C5 gene encoding human orhumanized C5 protein, wherein expression of the human C5 gene encodinghuman or humanized C5 protein is under control of rodent regulatoryelements at the endogenous rodent C5 locus. In one embodiment, therodent is a mouse. In one embodiment, the rodent is a rat.

In one embodiment, the human C5 gene encoding human or humanized C5protein comprises exon 2 through exon 41 of the human C5 gene.

In one embodiment, the rodent is a mouse that is incapable of expressinga mouse C5 protein.

In one embodiment, the rodent is a mouse that expresses a mouse C3protein encoded by an endogenous mouse C3 gene.

In one embodiment, the rodent is mouse that expresses a human orhumanized C3 protein.

In one embodiment, the rodent is a mouse that comprises a replacement atan endogenous mouse C3 locus of a mouse gene encoding C3 protein with ahuman C3 gene encoding a human or humanized C3 protein.

In one embodiment, expression of the human C3 gene encoding the human orhumanized C3 protein is under control of mouse regulatory elements atthe endogenous mouse C3 locus.

In one aspect, genetically modified rodents, e.g., mice or rats, areprovided comprising a replacement at an endogenous rodent C3 locus of arodent gene encoding C3 protein with a human C3 gene encoding human orhumanized C3 protein, wherein expression of the human C3 gene encodinghuman or humanized C3 protein is under control of rodent regulatoryelements at the endogenous rodent C3 locus. In one embodiment, therodent is a mouse. In one embodiment, the rodent is a rat.

In one embodiment, the human C3 gene encoding human or humanized C3protein comprises exon 1 through exon 41 of the human C3 gene.

In one embodiment, the human C3 gene encoding human or humanized C3protein comprises exon 2 through exon 41 of the human C3 gene.

In one embodiment, the rodent is a mouse that is incapable of expressinga mouse C3 protein.

In one embodiment, the rodent is a mouse that expresses a mouse C5protein encoded by an endogenous mouse C5 gene.

In one embodiment, the rodent is mouse that expresses a human orhumanized C5 protein.

In one embodiment, the rodent is a mouse that comprises a replacement atan endogenous mouse C5 locus of a mouse gene encoding C5 protein with ahuman C5 gene encoding a human or humanized C5 protein.

In one embodiment, expression of the human C5 gene encoding the human orhumanized C5 protein is under control of mouse regulatory elements atthe endogenous mouse C5 locus.

In one aspect, genetically modified rodents, e.g., a mouse or rat, areprovided that express a human or humanized C5 protein, wherein therodent that expresses a human or humanized C5 protein comprises a normalcomplement system, e.g., the levels of complement proteins in the blood,plasma or serum of the rodent expressing human or humanized C5 proteinare similar to the levels of complement proteins in the blood, plasma orserum of a rodent that expresses functional endogenous C5 protein. Inone embodiment, the rodent is a mouse. In one embodiment, the rodent isa rat.

In one aspect, genetically modified rodents, e.g., a mouse or rat, areprovided that express C5 protein from a human C5 gene, wherein therodent expresses human or humanized C5 protein in its serum. In oneembodiment, the rodent is a mouse. In one embodiment, the rodent is arat.

In one embodiment, the serum of the rodent that expresses a human orhumanized C5 protein has approximately the same level of C5 protein as arodent that expresses a functional, endogenous C5 protein, e.g., awild-type mouse or rat. In one embodiment, the rodent is a mouse. In oneembodiment, the rodent is a rat.

In one embodiment, the mouse expresses human or humanized C5 protein inserum at a concentration of at least about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%,190% or 200% of the level of C5 protein present in the serum of anage-matched mouse that expresses functional endogenous C5 protein, butdoes not comprise a replacement of an endogenous C5 gene, at anendogenous mouse C5 locus, with a human C5 gene.

In one embodiment, the mouse expresses human or humanized C5 protein inserum at a concentration of between about 10% and about 200%, betweenabout 20% and about 150%, or between about 30% and about 100% of thelevel of mouse C5 protein present in the serum of an age-matched mousethat expresses functional endogenous C5 protein, but does not comprise areplacement of an endogenous C5 gene, at an endogenous mouse C5 locus,with a human C5 gene.

In one embodiment, the mouse expresses human or humanized C5 protein inserum at a concentration of between about 10 μg/ml and about 150 μg/ml,between about 10 μg/ml and about 125 μg/ml, or between about 15 μg/mland about 100 μg/ml.

In one embodiment, the mouse expresses human or humanized C5 protein inserum at a concentration of at least about 10, 20, 30, 40, 50, 60, 70,80, 90, 100, 125 or 150 μg/ml.

In one aspect, genetically modified rodents, e.g., a mouse or rat, areprovided that express a human or humanized C3 protein, wherein therodent that expresses a human or humanized C3 protein comprises a normalcomplement system, i.e., the levels of complement proteins in the blood,plasma or serum of the rodent expressing human or humanized C3 proteinare similar to the levels of complement proteins in the blood, plasma orserum of a rodent that expresses functional endogenous C3 protein. Inone embodiment, the rodent is a mouse. In one embodiment, the rodent isa rat.

In one aspect, genetically modified rodents, e.g., a mouse or rat, areprovided that express C3 protein from a human C3 gene, wherein therodent expresses human or humanized C3 protein in its serum. In oneembodiment, the rodent is a mouse. In one embodiment, the rodent is arat.

In one embodiment, the serum of the rodent that expresses a human orhumanized C3 protein has approximately the same level of C3 protein as arodent that expresses a functional, endogenous C3 protein, e.g., awild-type mouse or rat. In one embodiment, the rodent is a mouse. In oneembodiment, the rodent is a rat.

In one embodiment, the mouse expresses human or humanized C3 protein(hC3) in serum at a concentration of at least about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%,180%, 190% or 200% of the level of C3 protein present in the serum of anage-matched mouse that expresses functional endogenous C3 protein, butdoes not comprise a replacement of an endogenous C3 gene, at anendogenous mouse C3 locus, with a human C3 gene.

In one embodiment, the mouse expresses human C3 protein in serum at aconcentration of between about 10% and about 200%, between about 20% andabout 150%, or between about 30% and about 100% of the level of mouse C3protein present in the serum of an age-matched mouse that expressesfunctional endogenous C3 protein, but does not comprise a replacement ofan endogenous C3 gene, at an endogenous mouse C3 locus, with a human C3gene.

In one embodiment, the mouse expresses human or humanized C3 protein inserum at a concentration of between about 100 μg/ml and about 1500μg/ml, between about 200 μg/ml and about 1250 μg/ml, or between about300 μg/ml and about 1000 μg/ml.

In one embodiment, the mouse expresses human or humanized C3 protein inserum at a concentration of at least about 100, 200, 300, 400, 500, 600,700, 800, 900, 1000, 1250 or 1500 μg/ml.

In one aspect, a genetically modified rodent is provided, comprising ahuman C5 gene comprising a replacement at an endogenous rodent C5 locusof a rodent gene encoding C5 protein with a human C5 gene encoding humanor humanized C5 protein, wherein expression of the human C5 geneencoding human or humanized C5 protein is under control of rodentregulatory elements or sequences at the endogenous rodent C5 locus, andwherein the rodent further comprises a human C3 gene comprising areplacement at an endogenous rodent C3 locus of a rodent gene encodingC3 protein with a human C3 gene encoding human or humanized C3 protein,wherein expression of the human C3 gene encoding human or humanized C3protein is under control of rodent regulatory elements or sequences atthe endogenous rodent C3 locus. In one embodiment, the rodent is amouse. In one embodiment, the rodent is a rat.

In one embodiment, the mouse is incapable of expressing a mouse C5protein and incapable of expressing a mouse C3 protein.

In one embodiment, the rodent regulatory elements or sequences at theendogenous rodent C5 locus and/or rodent C3 locus are from a mouse or arat.

In one embodiment, the rodent regulatory elements or sequences areendogenous rodent regulatory elements or sequences at the rodent C5locus and/or rodent C3 locus are from a mouse or a rat.

In one aspect, a non-human animal, e.g., a rodent, e.g., a mouse or rat,is provided that expresses human or humanized C5 and/or C3 proteins,wherein the non-human animal expresses human or humanized C5 and/or C3proteins from an endogenous non-human C5 locus and/or an endogenousnon-human C3 locus. In an embodiment, the non-human animal is a rodent.In an embodiment, the rodent is a mouse. In an embodiment, the rodent isa rat.

In one aspect, a genetically modified mouse is provided that expresseshuman or humanized C5 protein from an endogenous mouse C5 locus, whereinthe endogenous mouse C5 gene has been replaced, in whole or in part,with a human C5 gene.

In one embodiment, about 75.8 kb at the endogenous mouse C5 locus,including exons 2 through 42 and a 3′ untranslated sequence, is deletedand replaced with about 97 kb of human C5 gene sequence comprising exons2 through 41 of the human C5 gene. In a specific embodiment, the humanC5 gene comprises exons 2 through 42 of the human C5 gene of human BACCTD-2559119. In a specific embodiment, the C5 gene comprises mouse C5exon 1 and human C5 exons 2 through 42.

In one aspect, a genetically modified mouse is provided that comprises anucleotide sequence encoding a human or humanized C5 protein, whereinthe nucleotide sequence encoding the human or humanized C5 proteinreplaces, in whole or in part, an endogenous nucleotide sequenceencoding an endogenous mouse C5 protein.

In one aspect, a genetically modified mouse is provided that expresseshuman or humanized C3 protein from an endogenous mouse C3 locus, whereinthe endogenous mouse C3 gene has been replaced, in whole or in part,with a human C3 gene.

In one embodiment, a portion of the endogenous mouse C3 locus, including5′ regulatory elements upstream of exon 1 through exon 41, is deletedand replaced with a human C3 gene sequence comprising 5′ regulatoryelements upstream of exon 1 through exon 41 of the human C3 gene. In aspecific embodiment, the human C3 gene comprises the entire human C3coding region.

In one embodiment, a portion of the endogenous mouse C3 locus, includinga portion of intron 1 and exons 2 through 41, is deleted and replacedwith a human C3 gene sequence comprising a portion of intron 1 and exons2 through exon 41 of the human C3 gene. In a specific embodiment, the C3gene comprises mouse C3 exon 1 and human C3 exons 2 through 41.

In one aspect, a method is provided for making a humanized C5 rodent,comprising replacing a rodent C5 gene sequence encoding rodent C5protein with a human C5 gene sequence comprising one or more exons ofthe human C5 gene sequence encoding human or humanized C5 protein. Inone embodiment, the rodent is a mouse. In one embodiment, the rodent isa rat.

In one embodiment, the replacement is at an endogenous rodent C5 locusand the human C5 gene sequence comprising one or more exons of the humanC5 gene sequence encoding human or humanized C5 protein is operablylinked to rodent regulatory elements or sequences at the endogenousrodent C5 locus. In one embodiment, the rodent is a mouse. In oneembodiment, the rodent is a rat.

In one embodiment, the rodent regulatory elements or sequences arederived from a mouse. In one embodiment, the rodent regulatory elementsor sequences are derived from a rat.

In one embodiment, the rodent regulatory elements or sequences areendogenous rodent regulatory elements or sequences at the rodent C5locus. In one embodiment, the rodent is a mouse. In one embodiment, therodent is a rat.

In one embodiment, the human C5 gene sequence replacing the rodent C5gene sequence comprises at least one exon of the human C5 gene sequence.In other embodiments, the human C5 gene sequence replacing the rodent C5gene sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, or 40 exons of the human C5 genesequence. In one embodiment, the human C5 gene sequence replacing therodent C5 gene sequence comprises all 41 exons of the human C5 genesequence. In one embodiment, the rodent is a mouse. In one embodiment,the rodent is a rat.

In one embodiment, the replacement is at an endogenous rodent C5 locusand the human C5 gene sequence comprising one or more exons of the humanC5 gene sequence encoding human or humanized C5 protein is operablylinked endogenous rodent regulatory elements or sequences at theendogenous rodent C5 locus.

In one aspect, a method is provided for making a humanized C5 mouse,comprising replacing a mouse C5 gene sequence encoding mouse C5 proteinwith a human C5 gene sequence encoding human or humanized C5 protein.

In one embodiment, the replacement is at an endogenous mouse C5 locus,and the human C5 gene encoding human or humanized C5 protein is operablylinked to mouse regulatory elements or sequences at the endogenous mouseC5 locus.

In one embodiment, the replacement is at an endogenous mouse C5 locus,and the human C5 gene encoding human or humanized C5 protein is operablylinked to endogenous mouse regulatory elements or sequences at theendogenous mouse C5 locus.

In one aspect, a method is provided for making a humanized C3 rodent,comprising replacing a rodent C3 gene sequence encoding rodent C3protein with a human C3 gene sequence comprising one or more exons ofthe human C3 gene sequence encoding human or humanized C3 protein. Inone embodiment, the rodent is a mouse. In one embodiment, the rodent isa rat.

In one embodiment, the replacement is at an endogenous rodent C3 locusand the human C3 gene sequence comprising one or more exons of the humanC3 gene sequence encoding human or humanized C3 protein is operablylinked to rodent regulatory elements or sequences at the endogenousrodent C3 locus. In one embodiment, the rodent is a mouse. In oneembodiment, the rodent is a rat.

In one embodiment, the rodent regulatory elements or sequences arederived from a mouse. In one embodiment, the rodent regulatory elementsor sequences are derived from a rat.

In one embodiment, the rodent regulatory elements or sequences areendogenous rodent regulatory elements or sequences at the rodent C3locus. In one embodiment, the rodent is a mouse. In one embodiment, therodent is a rat.

In one embodiment, the human C3 gene sequence replacing the rodent C3gene sequence comprises at least one exon of the human C3 gene sequence.In other embodiments, the human C3 gene sequence replacing the rodent C3gene sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, or 40 exons of the human C3 genesequence. In one embodiment, the human C3 gene sequence replacing therodent C5 gene sequence comprises all 41 exons of the human C3 genesequence. In one embodiment, the rodent is a mouse. In one embodiment,the rodent is a rat.

In one embodiment, the replacement is at an endogenous rodent C3 locusand the human C3 gene sequence comprising one or more exons of the humanC3 gene sequence encoding human or humanized C3 protein is operablylinked endogenous rodent regulatory elements or sequences at theendogenous rodent C3 locus.

In one aspect, a method is provided for making a humanized C3 mouse,comprising replacing a mouse C3 gene sequence encoding mouse C3 proteinwith a human C3 gene sequence encoding human or humanized C3 protein.

In one embodiment, the replacement is at an endogenous mouse C3 locus,and the human C3 gene encoding human or humanized C3 protein is operablylinked to endogenous mouse regulatory elements or sequences at theendogenous mouse C3 locus.

In one embodiment, the replacement is at an endogenous mouse C3 locus,and the human C3 gene encoding human or humanized C3 protein is operablylinked to mouse regulatory elements or sequences at the endogenous mouseC3 locus.

In various aspects, the genetically modified non-human animals, e.g.,rodents, e.g., mice or rats, described herein comprise the geneticmodifications in their germ-line.

In one aspect, a non-human animal, e.g., rodent, e.g., a mouse or rat,embryo comprising a genetic modification as described herein isprovided.

In one aspect, a non-human animal, e.g., rodent, e.g. a mouse or rat,host embryo is provided that comprises a donor cell that comprises agenetic modification as described herein.

In one aspect, a pluripotent or totipotent non-human animal, e.g.,rodent, e.g., mouse or rat, cell comprising a genetic modification asdescribed herein is provided. In one embodiment, the cell is a rodentcell. In one embodiment, the cell is a mouse cell. In one embodiment,the cell is a rodent ES cell. In one embodiment, the cell is a mouse EScell.

In one aspect, a non-human animal, e.g., rodent, e.g., mouse or rat, eggis provided, wherein the non-human animal egg comprises an ectopicnon-human animal chromosome, wherein the ectopic non-human animalchromosome comprises a genetic modification as described herein. In oneembodiment, the non-human animal is a rodent. In one embodiment, therodent is a mouse. In one embodiment, the rodent is a rat.

In one aspect, the mouse embryo, egg, or cell that is geneticallymodified to comprise a human C5 gene or human C3 gene is of a mouse thatis of a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa,C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10,C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In another embodiment, themouse is a 129 strain selected from the group consisting of a strainthat is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm),129S2, 129S4, 129S5, 12959/SvEvH, 129S6 (129/SvEvTac), 129S7, 129S8,129T1, 129T2 (see, e.g., Festing et al. (1999) Revised nomenclature forstrain 129 mice, Mammalian Genome 10:836, see also, Auerbach et al(2000) Establishment and Chimera Analysis of 129/SvEv- andC57BL/6-Derived Mouse Embryonic Stem Cell Lines). In a specificembodiment, the genetically modified mouse is a mix of an aforementioned129 strain and an aforementioned C57BL/6 strain. In another specificembodiment, the mouse is a mix of aforementioned 129 strains, or a mixof aforementioned BL/6 strains. In a specific embodiment, the 129 strainof the mix is a 129S6 (129/SvEvTac) strain. In another embodiment, themouse is a BALB strain, e.g., BALB/c strain. In yet another embodiment,the mouse is a mix of a BALB strain and another aforementioned strain.In one embodiment, the mouse is Swiss or Swiss Webster mouse.

In a further aspect, provided herein are methods for identifying acompound capable of modulating complement activation comprisingadministering the compound to any of the non-human animals, e.g., arodent, e.g., a mouse or rat disclosed and described herein; andassaying if complement activation in the rodent is modulated, therebyidentifying a compound capable of modulating complement activation. Inone embodiment, the compound is a small molecule chemical compound, anantibody, a protein, an inhibitory nucleic acid, or any combinationthereof. In another embodiment of any of the embodiments disclosedherein, the compound modulates complement activation by increasingcomplement activity. In another embodiment of any of the embodimentsdisclosed herein, the compound modulates complement activation bydecreasing complement activity. In another embodiment of any of theembodiments disclosed herein, the serum of the rodent is assayed todetermine if complement activation in the rodent is modulated. In afurther embodiment, the assay is a CH₅₀ complement screening assay.

Each of the aspects and embodiments described herein are capable ofbeing used together, unless excluded either explicitly or clearly fromthe context of the embodiment or aspect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides an illustration, not to scale, of the mouse (top) andhuman (bottom) C5 genomic loci. The regions of the mouse and human C5genes that are deleted and replaced, respectively, to generate humanizedC5 mice are indicated.

FIG. 2A provides an illustration, not to scale, of the mouse (mC3) andhumanized (hC3) C3 genomic loci. The mouse C3 gene spanning the 5′regulatory elements and the coding region from exon 1 through exon 41are deleted and replaced by the 5′ regulatory elements and the codingregion from exon 1 through exon 41 of the human C3 gene and a loxP site,as indicated by a bold line and an arrow, respectively. FIG. 2B providesan illustration, not to scale, of the mouse (mC3) and humanized (hC3) C3genomic loci. The mouse C3 gene spanning a portion of intron 1 and thecoding region from exon 2 through exon 41 are deleted and replaced by aportion of intron 1 and the coding region from exon 2 through exon 41 ofthe human C5 gene and a loxP site, as indicated by a bold line and anarrow, respectively.

FIG. 3 shows that mouse complement has lower activity in a hemolyticassay than human complement.

FIG. 4 shows that complement hemolytic activity is blocked byspecies-specific anti-C5 monoclonal antibodies in a species-specificmanner.

FIG. 5A and FIG. 5B depict graphs showing that humanized C5 mice arefunctional mouse C5 knockouts. Addition of human C5 or C3 proteinreconstitutes hemolytic activity in C5 or C3-depleted human serum,respectively, as depicted in FIG. 5A. Mouse, but not human, C5 proteinreconstitutes hemolytic activity in C5^(−/−) and humanized C5 mousesera, depicted in FIG. 5B. FIG. 5C is a bar graph depicting levels ofhuman C5 protein in humanized C5 mouse serum that are three times lowerthan in human serum. FIG. 5D is a bar graph depicting that pre-dosecomplement C5 levels in humanized C5 mice vary between males andfemales. A range of human C5, from 20-100 μg/mL is present in humanizedC5 mouse serum.

FIG. 6 shows that addition of human C3 and C5 proteins, but not human C5protein alone, reconstitutes hemolytic activity in C5^(−/−) mouse serum.

FIG. 7 shows that humanized C5 mouse serum requires addition of human C3protein to achieve wild-type levels of mouse hemolytic activity.

FIG. 8A depicts graphs showing the results of a hemolysis assayperformed on serum samples from humanized C5 mice exposed to theanti-human C5 antibody (C5Ab) using 400 μg/mL human C3 protein. FIG. 8Bdepicts graphs showing the results of a hemolysis assay performed onserum samples from humanized C5 mice exposed to the anti-human C5antibody (C5Ab) using 800 μg/mL human C3 protein.

FIG. 9A depicts a graph showing the results of a pharmacokinetic assaymeasuring anti-human C5 antibody levels as a function of timepost-injection for all animals in the study. FIG. 9B depicts graphsshowing the results of a pharmacokinetic assay measuring anti-human C5antibody levels as a function of time post-injection for female (top)and male (bottom) animals.

FIG. 10A shows that adding human C3 protein to wild type mouse serum canenhance hemolytic function but not to the same extent observed in normalhuman serum (for each data point from mice, serum was mixed from 3animals). FIG. 10B shows that hemolytic function can be restored inhumanized C5^(hu/hu) mice when supplemented with human C3 protein.

FIG. 11 shows that adding human C3 protein to serum derived from C3knockout mice can rescue hemolytic function (for each data point frommice, serum was mixed from 3 animals).

FIG. 12 shows that adding human C3 protein only to C5 knockout mouseserum cannot rescue hemolytic function (for each data point from mice,serum was mixed from 3 animals).

DETAILED DESCRIPTION

The complement system is an essential component of the innate immunesystem and plays a critical role as a defense mechanism against invadingpathogens, primes adaptive immune responses, and helps remove immunecomplexes and apoptotic cells. While the complement system plays acritical role in many protective immune functions, complement activationis a significant mediator of tissue damage in a wide range of autoimmuneand inflammatory disease processes.

The invention described herein provides, inter alia, non-human animalsthat express human or humanized C5 and/or C3 proteins in serum atconcentrations similar to wild type expression of endogenous C5 and/orC3 proteins in non-human animals. The successful engineering ofnon-human animals capable of expressing human or humanized C5 and/or C3proteins provides a needed and useful tool for recapitulating the humancomplement system in laboratory animals amenable to large scale andhigh-throughput drug screening assays. Also provided herein are methodsfor using the non-human animals (such as rodents) described herein foridentifying compounds capable of modulating complement activation. Themethods are based, in part, on the inventors' discovery that whilecomplement activity of C5 protein is species-specific (i.e., human C5protein is unable to substitute for mouse C5 protein), the complementactivity of human C3 protein is able to substitute for mouse C3 proteinin a mouse serum complement activity.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,cell biology, biochemistry, nucleic acid chemistry, and immunology,which are well known to those skilled in the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, fourth edition (Sambrook et al., 2012) and MolecularCloning: A Laboratory Manual, third edition (Sambrook and Russel, 2001),(jointly referred to herein as “Sambrook”); Current Protocols inMolecular Biology (F. M. Ausubel et al., eds., 1987, includingsupplements through 2014); PCR: The Polymerase Chain Reaction, (Mulliset al., eds., 1994); Antibodies: A Laboratory Manual, Second edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(Greenfield, ed., 2014), Beaucage et al. eds., Current Protocols inNucleic Acid Chemistry, John Wiley & Sons, Inc., New York, 2000,(including supplements through 2014) and Gene Transfer and Expression inMammalian Cells (Makrides, ed., Elsevier Sciences B.V., Amsterdam,2003).

Definitions

As used herein, the term “protein” includes polypeptides, peptides,fragments of polypeptides, and fusion polypeptides.

As used herein, a “nucleic acid” refers to two or moredeoxyribonucleotides and/or ribonucleotides covalently joined togetherin either single or double-stranded form.

By “operably linked” is meant a functional linkage between a nucleicacid expression control sequence (such as a promoter) and a secondnucleic acid sequence, wherein the expression control sequence directstranscription of the nucleic acid corresponding to the second sequence.

The term “replacement” in reference to gene replacement refers toplacing exogenous genetic material at an endogenous genetic locus,thereby replacing all or a portion of the endogenous gene with anorthologous or homologous nucleic acid sequence. In one instance, anendogenous non-human gene or fragment thereof is replaced with acorresponding human gene or fragment thereof. A corresponding human geneor fragment thereof is a human gene or fragment that is an ortholog of,a homolog of, or is substantially identical or the same in structureand/or function, as the endogenous non-human gene or fragment thereofthat is replaced. As demonstrated in the Examples below, nucleotidesequences of endogenous non-human (for example rodent, such as, mouse)C3 and/or C5 gene loci were replaced by nucleotide sequencescorresponding to human C3 and/or C5 gene loci. In another embodiment, agene replacement can occur when an endogenous gene is deleted orrendered nonfunctional (such as by the insertion of a missense mutationor a premature stop codon) and a corresponding human gene or fragmentthereof is inserted into the germline at a separate location.

The term “humanized” as used in the phrases “humanized C3 allele” or“humanized C5 allele,” or “humanized C3 gene,” or “humanized C5 gene”includes, but is not limited to, embodiments wherein all or a portion ofan endogenous non-human C3 and/or C5 gene or allele is replaced by acorresponding portion of the human C3 and/or C5 gene or allele. Forexample, in some embodiments, the term “humanized” refers to thecomplete replacement of the coding region (e.g., the exons) of theendogenous non-human C3 and/or C5 gene or allele with the correspondingcoding region of the human C3 and/or C5 gene or allele, while theendogenous non-coding region(s) (such as, but not limited to, thepromoter, the 5′ and/or 3′ untranslated region(s), enhancer elements,etc.) of the non-human animal is not replaced. In some embodiments, thenon-human animal is a rodent, such as a rat or mouse.

A “humanized protein” includes, but is not limited to, embodimentswherein all or a portion of the encoded endogenous non-human C3 and/orC5 protein is replaced by the corresponding portion of the human C3and/or C5 protein. In some embodiments, a “humanized protein” can beencoded by a humanized C3 and/or C5 gene or allele but still encode afully human C3 and/or C5 protein (such as, but not limited to, thesituation wherein all of the coding regions (e.g., the exons) of theendogenous non-human C3 and/or C5 gene or allele are replaced by thecorresponding coding regions of the human C3 and/or C5 gene or allelebut the endogenous non-coding region(s) (such as, but not limited to,the promoter, the 5′ and/or 3′ untranslated region(s), enhancerelements, etc.) of the non-human animal is not replaced). In someembodiments, the non-human animal is a rodent, such as a rat or mouse.

“Modulates,” as used herein, refers to the ability of a compound toafter the activity of the complement system. In one embodiment,modulation of the complement system by a compound refers to decreasingcomplement activation in a subject (for example, a rodent, such as amouse). In another embodiment, modulation of the complement system by acompound refers to increasing complement-mediated activity in a subject.

As used herein, the term “rodent” refers to any member of the orderRodentia. Examples of rodents include, without limitation, mice, rats,squirrels, prairie dogs, porcupines, beavers, guinea pigs, and hamsters.In one embodiment, a rodent is a rat. In another embodiment, a rodent isa mouse.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains.

As used herein, the singular terms “a,” “any” and “the” include theplural reference unless the context clearly indicates otherwise.

It is intended that every maximum numerical limitation given throughoutthis specification includes every lower numerical limitation, as if suchlower numerical limitations were expressly written herein. Every minimumnumerical limitation given throughout this specification will includeevery higher numerical limitation, as if such higher numericallimitations were expressly written herein. Every numerical range giventhroughout this specification will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein.

Complement System

Complement, an essential component of the immune system, consists ofmore than 30 serum and cellular proteins that are involved in two linkedbiochemical cascades, the classical and alternative pathways. Complementfunctions to assist the immune system to destroy invading microorganismsand maintain tissue homeostasis.

However, excessive or unregulated activation of complement contributesto tissue damage, and is associated with a variety of variety of humandiseases, disorders and conditions that are associated with complementactivation, for example, ocular inflammatory and retinal degenerativediseases (see Makrides (1998) Therapeutic inhibition of the complementsystem, Pharmacological Reviews 50(1):59-87; and Mollnes et al. (2006)Strategies of therapeutic complement inhibition, Molecular Immunology43; 107-121). Other diseases or conditions known to be associated withaberrant complement activation include, without limitation, allergicasthma and the accompanying airway inflammation and airwayhyperresponsiveness (“AHR”), chronic obstructive pulmonary disease(“COPD”), allergic bronchopulmonary aspergillosis, hypersensitivitypneumonia, eosinophilic pneumonia, emphysema, bronchitis, allergicbronchitis, bronchiectasis, cystic fibrosis, tuberculosis,hypersensitivity pneumonitis, occupational asthma, sarcoid, reactiveairway disease syndrome, interstitial lung disease, hyper-eosinophilicsyndrome, rhinitis, sinusitis, exercise-induced asthma,pollution-induced asthma, cough variant asthma, parasitic lung disease,respiratory syncytial virus (“RSV”) infection, parainfluenza virus(“PIV”) infection, rhinovirus (“RV”) infection and adenovirus infection,and ischemia-reperfusion injury (see, e.g., U.S. Patent ApplicationPublication No. 2005/0260198, which is incorporated herein byreference).

Complement C5 Gene and Protein

The C5 gene encodes the serum complement C5 protein, which plays animportant role in inflammatory and cell killing processes. Mature C5protein is comprised of α and β chains that are linked by a disulfidebond. The C5 protein is cleaved by a convertase, resulting in anactivation peptide, C5a, an anaphylatoxin derived from the αpolypeptide, which is largely pro-inflammatory, and C5b, amacromolecular cleavage product derived from the β polypeptide, whichforms a complex with other complement components to form a membraneattack complex (MAC), which is involved in osmotic lysis of targetcells.

Human C5. Official symbol: C5; NCBI Gene ID: 727; Primary source:HGNC:1331; RefSeq mRNA ID: NM_001735.2; UniProt ID: P01031; Genomicassembly: GRCh38; Location: chr9:120,952,335-121,050,275-strand.

The human C5 gene is located on chromosome 9, at 9q33-q34. The human C5gene has 41 exons and encodes a precursor polypeptide of 1676 aminoacids in length, including an 18 amino acid signal peptide, a 655 aminoacid β chain and a 999 amino acid α chain. During complement activationthe α chain is cleaved, thereby generating a 74 amino acid C5a, which isa potent pro-inflammatory anaphylatoxin.

C5 deficiency in humans is associated with increased susceptibility tosevere recurrent infections.

The C5 gene is conserved between several species, including primates,e.g., chimpanzee, Rhesus monkey, other mammals, e.g., dog, cow, rodent,e.g., mouse, chicken, zebrafish and frog.

Mouse C5. Official symbol: Hc; NCBI Gene ID: 15139; Primary source:MGI:96031; RefSeq mRNA ID: NM_010406.2; UniProt ID: P06684; Genomicassembly: GRCm38; Location: chr2:34,983,331-35,061,449-strand.

The mouse C5 gene is located on chromosome 2, at 2 23.22 cM. The mouseC5 gene has 42 exons and encodes a precursor polypeptide of 1680 aminoacids in length, including an 18 amino acid signal peptide, a 656 aminoacid β chain and a 1002 amino acid α chain. During complement activationthe α chain is cleaved, thereby generating a 77 amino acid C5a, which isa potent pro-inflammatory anaphylatoxin.

C5 deficiency is observed in several common strains of laboratory micewhich have a common 2 base pair deletion near the 5′ end of the cDNAthat results in inability of C5 to be secreted; thus, these mice arefunctionally C5 deficient, i.e., C5 knockouts (6 such strains are A/HeJ,AKR/J, DBA/2J, NZB/B1 NJ, SWR/J, and B10.D2/oSnJ) (see, e.g., Wetzel etal. (1990) Deficiency of the murine fifth complement component (C5): a2-base pair deletion in a 5′ exon, J Biol Chem 265:2435-2440). C5knockout mice can also be generated following standard procedures knownin the art.

Complement C3 Gene and Protein

The C3 gene encodes the serum complement protein C3, which plays acentral role in the activation of the classical and alternativecomplement activation pathways.

The human C3 gene is located on chromosome 19 at 19p13.3-p13.2. Thehuman C3 gene has 41 exons and encodes a precursor polypeptide of 1663amino acids, including a 22 amino acid signal peptide, a 645 amino acidβ chain and a 992 amino acid α chain. During complement activation the αchain is cleaved, thereby generating 9 different peptides, including a77 amino acid C5a, which is a potent pro-inflammatory anaphylatoxin.

C3 deficiency in humans is associated with increased susceptibility tobacterial infections.

The C3 gene is conserved between several species, including primates,e.g., chimpanzee, Rhesus monkey, other mammals, e.g., dog, cow, rodent,e.g., mouse, chicken, zebrafish and frog.

The mouse C3 gene is located on chromosome 17 at 17 29.72 cM19. Themouse C3 gene has 41 exons and encodes a precursor polypeptide of 1663amino acids, including a 24 amino acid signal peptide, a 642 amino acidβ chain and a 993 amino acid α chain. During complement activation the αchain is cleaved, thereby generating 9 different peptides, including a78 amino acid C3a, which is a potent pro-inflammatory anaphylatoxin.

C3 knockout mice have been generated following standard procedures knownin the art (see, e.g., Drouin et al. (2001) Cutting edge: the absence of3 demonstrates a role for complement in Th2 effector functions in amurine model of pulmonary allergy, J Immunology 167:4141-4144).

Species Specificity of C5 and C3 Complement Proteins

Shown herein, humanized C5 mice are functionally C5 deficient because atleast some of the endogenous complement proteins displayspecies-specificity, i.e., the endogenous mouse complement proteins areunable to functionally interact with the human C5 protein. Serumobtained from humanized C5 mice or C5 knockout mice were tested forcomplement activity; no complement hemolytic activity was observedunless both human C3 and human C5 proteins were added.

Also shown herein, addition of human C3 to sera obtained from C3knockout mice was able to rescue hemolytic function; complement activitywas observed in C3 knockout mice when human C3 protein was added toserum.

The complement activity of C5 protein is species-specific, i.e., humanC5 protein is unable to substitute for mouse C5 protein in a mouse serumcomplement activity (i.e., hemolysis) assay.

The complement activity of C3 protein, in contrast does not appear to besimilarly species-specific, i.e., human C3 protein is able to substitutefor mouse C3 protein in a mouse serum complement activity (i.e.,hemolysis) assay.

Therapeutic Inhibition of the Complement System

Two targets for therapeutic complement inhibition are the complementactivation peptides C5a and C3a, which are generated upon complementactivation by enzymatic cleavage in both the classical and alternativepathways from complement proteins C5 and C3, respectively (see Markides(1998); Mollnes et al. (2006)).

For example, blocking C5 cleavage by antibodies, for example, antibodiesdisclosed in U.S. Pat. No. 6,355,245, is a possible therapeutic strategyfor complement inhibition in diseases associated with complementactivation.

Species Specificity of Human C5 and C3 Inhibitors

Candidate therapeutic molecules that target the complement proteins C5or C3 are typically evaluated for pharmacokinetics (PK) andpharmacodynamics (PK) in non-human animals, e.g., rodents, e.g., mice orrats. Such therapeutic molecules are also tested for in vivo therapeuticefficacy in non-human animal, e.g., rodent, e.g., mouse or rat, modelsof human diseases, disorders and conditions associated with complementactivation.

However, therapeutic molecules, which are specific for the humancomplement proteins C5 or C3, i.e., human-specific C5 or C3 inhibitors,cannot be adequately evaluated for PD or in vivo therapeutic efficacy inrodents, in particular mice, because the targets of these therapeuticmolecules are missing. This problem is not overcome using transgenicnon-human animals, e.g., rodents, e.g., mice or rats, expressing humanC5 or C3 complement proteins because of the above-mentioned speciesspecificity of these proteins.

Accordingly, to assess the PD and in vivo therapeutic efficacy of ahuman-specific C5 or C3 protein antagonist or inhibitor in non-humananimals, e.g., rodents, e.g., mice or rats, it is desirable to replacethe endogenous C5 and/or C3 proteins with human C5 and/or C3 proteins.Moreover, in order to avoid potential problems of over- orunder-expression of the human C5 and/or C3 proteins, it is desirable toinsert the human C5 and/or C3 genes into the genome of the non-humananimals, e.g., rodents, e.g., mice or rats, at the endogenous C5 and/orC3 gene loci, and to express the human C5 and/or C3 proteins innon-human animals, e.g., rodents, e.g., mice or rats, under the control,at least in part, of the endogenous C5 and/or C3 regulatory elements.

Creation of Humanized C3 and/or C5 Non-human Animals

Methods for generating the humanized C3 and/or C5 animals of the presentinvention are well known in the art (see, generally, Gene Targeting: APractical Approach, Joyner, ed., Oxford University Press, Inc. (2000)).In one embodiment, generation of the mice may optionally involvedisruption of murine C3 and/or C5 genes and introduction of the geneencoding human or humanized C3 and/or C5 into the murine genome. In oneembodiment, the introduction of the gene encoding human or humanized C3and/or C5 is at the same location as the endogenous murine C3 and/or C5genes.

The transgenic non-human animals of the invention can be produced byintroducing transgenes into the germline of the animal. Embryonic targetcells at various developmental stages can be used to introducetransgenes. Different methods are used depending on the stage ofdevelopment of the embryonic target cell. The specific line(s) of anyanimal used to practice this invention are selected for general goodhealth, good embryo yields, good pronuclear visibility in the embryo,and good reproductive fitness. When transgenic mice are to be produced,strains such as C57BL/6 or C57BL/6×DBA/2 F₁, or FVB lines are often used(obtained commercially from Charles River Labs, Boston, Mass., TheJackson Laboratory, Bar Harbor, Me., or Taconic Labs.).

Introduction of the transgene into the embryo can be accomplished by anymeans known in the art such as, for example, microinjection,electroporation, or lipofection. For example, the transgene(s) can beintroduced into a mammal by microinjection of the construct into thepronuclei of the fertilized mammalian egg(s) to cause one or more copiesof the construct to be retained in the cells of the developingmammal(s). Following introduction of the transgene construct into thefertilized egg, the egg may be incubated in vitro for varying amounts oftime, or reimplanted into the surrogate host, or both. In vitroincubation to maturity is within the scope of this invention. One commonmethod is to incubate the embryos in vitro for about 1-7 days, dependingon the species, and then reimplant them into the surrogate host.

Reimplantation is accomplished using standard methods. Usually, thesurrogate host is anesthetized, and the embryos are inserted into theoviduct. The number of embryos implanted into a particular host willvary by species, but will usually be comparable to the number of offspring the species naturally produces.

Retroviral infection can also be used to introduce transgene into anon-human animal. The developing non-human embryo can be cultured invitro to the blastocyst stage. During this time, the blastomeres can betargets for retroviral infection (Jaenich, R. (1976) PNAS 73:1260-1264).Efficient infection of the blastomeres is obtained by enzymatictreatment to remove the zona pellucida (Manipulating the Mouse Embryo,Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor,1986). The viral vector system used to introduce the transgene istypically a replication-defective retrovirus carrying the transgene(Jahner et al. (1985) PNAS 82:6927-6931; Van der Putten et al. (1985)PNAS 82:6148-6152).

A third type of target cell for transgene introduction is the embryonicstem (ES) cell. Transgenes can be efficiently introduced into the EScells by DNA transfection or by retrovirus-mediated transduction. Suchtransformed ES cells can thereafter be combined with blastocysts from anon-human animal. The ES cells thereafter colonize the embryo andcontribute to the germ line of the resulting chimeric animal.

Transgenic animals comprising humanized C3 and/or C5 can be crossed withother animals. A manner of preparation is to generate a series ofmammals, each containing one of the desired constructs or transgenes.Such mammals are bred together through a series of crosses, backcrossesand selections, to ultimately generate a single mammal containing alldesired constructs and/or transgenes, where the mammal is otherwisecongenic (genetically identical) to the wild type except for thepresence of the desired constructs and/or transgene(s). In oneembodiment, a mouse comprising a human or humanized C3 and/or C5 gene isproduced in this manner.

Typically, crossing and backcrossing is accomplished by mating siblingsor a parental strain with an offspring, depending on the goal of eachparticular step in the breeding process. In certain cases, it may benecessary to generate a large number of offspring in order to generate asingle offspring that contains each of the knockout constructs and/ortransgenes in the proper chromosomal location. In addition, it may benecessary to cross or backcross over several generations to ultimatelyobtain the desired genotype.

Use of Humanized C3 and/or C5 Non-human Animals for IdentifyingCompounds Capable of Modulating the Complement System

In some aspects, provided herein are methods for identifying a candidatetherapeutic molecule (i.e. a compound) capable of modulating complementactivation. The method utilizes any of the humanized C3 and/or C5rodents (for example, mice or rats) disclosed herein. In someembodiments, candidate compounds are administered directly to therodents following experimental induction of complement activation (forexample, in a kidney ischemia/reperfusion model) and the effects of saidcompounds with respect to their ability to modulate the complementsystem are assessed. In other embodiments, candidate compounds arecontacted with serum obtained from these animals and complement activityis assessed using any commonly used in vitro assessment technique (suchas, but not limited to CH₅₀ assays).

In some embodiments, the candidate compound can modulate complementactivation by reducing, decreasing, or inhibiting complement activation.The compound may reduce complement activation in any of the rodentsdisclosed herein by any of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,26% 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74.%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% in comparison to control rodents that arenot treated with the candidate compound.

In another embodiment, the candidate compound can reduce, decrease, orinhibit complement activation for up to 1 hour, 2 hours, 3 hours, 4hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10, hours, 11,hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48hours, 54 hours, 60 hours, 66 hours, 72 hours, 4 days, 5 days, 6 days, 7days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, orthree weeks, inclusive (including any periods of time in between thesevalues).

The candidate compound can further modulate complement activation inother embodiments by increasing, amplifying or activating complementactivity. The compound may increase complement activation in any of therodents disclosed herein by any of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% in comparison to control rodentsthat are not treated with the candidate compound.

In another embodiment, the candidate compound can increase, amplify oractivate complement activity for up to 1 hour, 2 hours, 3 hours, 4hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10, hours, 11,hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48hours, 54 hours, 60 hours, 66 hours, 72 hours, 4 days, 5 days, 6 days, 7days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, orthree weeks, inclusive (including any periods of time in between thesevalues).

Candidate compounds can be, without limitation, small molecule chemicalcompounds, antibodies, proteins, inhibitory nucleic acids, or anycombination thereof.

1. Antibodies

In some aspects, the candidate compound binds (such as preferentiallybinds) to a complement protein (such as, but not limited to, C5 or C3(e.g. C3a)) and is an antibody. In some embodiments, the antibodies areC5 and/or C3 antagonists and can decrease complement activation. Inother embodiments, the antibodies are C5 and/or C3 agonists and canincrease complement activation.

Variants of antibodies can also be made based on information known inthe art, without substantially affecting the activity of antibody. Forexample, antibody variants can have at least one amino acid residue inthe antibody molecule replaced by a different residue. For antibodies,the sites of greatest interest for substitutional mutagenesis generallyinclude the hypervariable regions, but framework region (FR) alterationsare also contemplated.

For antibodies, one type of substitutional variant involves substitutingone or more hypervariable region residues of a parent antibody (e.g. ahumanized or human antibody). Generally, the resulting variant(s)selected for further development will have improved biologicalproperties relative to the parent antibody from which they aregenerated. A convenient way for generating such substitutional variantsinvolves affinity maturation using phage display. Briefly, severalhypervariable region sites (e.g. 6-7 sites) are mutated to generate allpossible amino acid substitutions at each site. The antibodies thusgenerated are displayed from filamentous phage particles as fusions tothe gene III product of M13 packaged within each particle. Thephage-displayed variants are then screened for their biological activity(e.g. binding affinity) as herein disclosed. In order to identifycandidate hypervariable region sites for modification, alanine scanningmutagenesis can be performed to identify hypervariable region residuescontributing significantly to antigen binding.

Nucleic acid molecules encoding amino acid sequence variants of theantibody can be prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications inan Fc region of the immunoglobulin polypeptides of the invention,thereby generating a Fc region variant. The Fc region variant maycomprise a human Fc region sequence (e.g. a human IgGI, IgG2, IgG3 orIgG4 Fc region) comprising an amino acid modification (e.g. asubstitution) at one or more amino acid positions including that of ahinge cysteine.

Fc region variants with altered (i.e. improved or diminished) Clqbinding and/or Complement Dependent Cytotoxicity (CDC) are described inInternational Patent Application Publication No.: W099/51642(incorporated herein by reference). Such variants may comprise an aminoacid substitution at one or more of amino acid positions of the Fcregion. See, also, Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat.Nos. 5,648,260; 5,624,821; and International Patent ApplicationPublication No.: W094/29351 concerning Fc region variants, thedisclosures of each of which are incorporated by reference herein.

2. Non-Antibody Binding Polypeptides

In some aspects, the candidate compound binds (such as preferentiallybinds) to a complement protein (such as, C5 or C3 (e.g. C3a)) and is anon-antibody binding polypeptide. In some embodiments, the non-antibodybinding polypeptide is a C5 and/or C3 antagonist and can decreasecomplement activation. In other embodiments, the non-antibody bindingpolypeptide is a C5 and/or C3 agonist and can increase complementactivation.

Binding polypeptides may be chemically synthesized using knownpolypeptide synthesis methodology or may be prepared and purified usingrecombinant technology. Binding polypeptides are usually at least about5 amino acids in length, alternatively at least about 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100 amino acids in length or more, wherein such binding polypeptidesthat are capable of binding to a target, such as any of the complementproteins (e.g. C5 or C3) discussed herein.

Binding polypeptides may be identified without undue experimentationusing well known techniques. In this regard, it is noted that techniquesfor screening polypeptide libraries for binding polypeptides that arecapable of binding to a polypeptide target are well known in the art(see, e.g., U.S. Pat. Nos. 5,556,762, 5,750,373, 4,708,871, 4,833,092,5,223,409, 5,403,484, 5,571,689, 5,663,143; PCT Application PublicationNos. WO 84/03506 and WO84/03564; Geysen et al, Proc. Natl. Acad. Sci.U.S.A., 81:3998-4002 (1984); Geysen et al, Proc. Natl. Acad. Sci.U.S.A., 82: 178-182 (1985); Geysen et al., J. Immunol. Meth, 102:259-274(1987); Clackson, T. et al., (1991) Nature, 352: 624; Kang, A. S. etal., (1991) Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991)Current Opin. Biotechnol, 2:668, the disclosures of each of which areincorporated by reference herein.

Methods for generating peptide libraries and screening these librariesare also disclosed in U.S. Pat. Nos. 5,723,286, 5,432,018, 5,580,717,5,427,908, 5,498,530, 5,770,434, 5,734,018, 5,698,426, 5,763,192, and5,723,323, the disclosures of each of which are incorporated byreference herein.

Binding polypeptides can be modified to enhance their inhibitory and/ortherapeutic effect (including, for example, enhanced affinity, improvedpharmacokinetic properties such as half-life, stability, and clearancerate, reduced toxicity, etc.). Such modifications include, withoutlimitation, glycosylation, pegylation, substitution with non-naturallyoccurring but functionally equivalent amino acid, linking groups, etc.

3. Small Molecules

In some aspects, the candidate compound binds (such as preferentiallybinds) to a complement protein (such as, C5 or C3 (e.g. C3a)) and is asmall molecule. In some embodiments, the small molecule is a C5 and/orC3 antagonist and can decrease complement activation. In otherembodiments, the small molecule is a C5 and/or C3 agonist and canincrease complement activation.

Small molecules are preferably organic molecules other than bindingpolypeptides or antibodies as defined herein. Organic small moleculesmay be identified and chemically synthesized using known methodology(see, e.g., PCT Application Publication Nos. WO 00/00823 and WO00/39585). Organic small molecules are usually less than about 2000Daltons in size, alternatively less than about 1500, 750, 500, 250 or200 Daltons in size, wherein such organic small molecules that arecapable of binding to a polypeptide as described herein may beidentified without undue experimentation using well known techniques. Inthis regard, it is noted that techniques for screening organic smallmolecule libraries for molecules that are capable of binding to apolypeptide target are well known in the art (see, e.g., PCT ApplicationPublication Nos. WO 00/00823 and WO 00/39585).

Organic small molecules may be, for example, aldehydes, ketones, oximes,hydrazones, semicarbazones, carbazides, primary amines, secondaryamines, tertiary amines, N-substituted hydrazines, hydrazides, alcohols,ethers, thiols, thioethers, disulfides, carboxylic acids, esters,amides, ureas, carbamates, carbonates, ketals, thioketals, acetals,thioacetals, aryl halides, aryl sulfonates, alkyl halides, alkylsulfonates, aromatic compounds, heterocyclic compounds, anilines,alkenes, alkynes, diols, amino alcohols, oxazolidines, oxazolines,thiazolidines, thiazolines, enamines, sulfonamides, epoxides,aziridines, isocyanates, sulfonyl chlorides, diazo compounds, acidchlorides, or the like.

In some aspects, the small molecule chemical compound is a component ofa combinatorial chemical library. Combinatorial chemical libraries are acollection of multiple species of chemical compounds comprised ofsmaller subunits or monomers. Combinatorial libraries come in a varietyof sizes, ranging from a few hundred to many hundreds of thousanddifferent species of chemical compounds. There are also a variety oflibrary types, including oligomeric and polymeric libraries comprised ofcompounds such as carbohydrates, oligonucleotides, and small organicmolecules, etc. Such libraries have a variety of uses, such asimmobilization and chromatographic separation of chemical compounds, aswell as uses for identifying and characterizing ligands capable ofbinding a target molecule (for example, C5 and/or C3) or mediating abiological activity of interest (such as, but not limited to, inhibitionor activation of complement activity).

Various techniques for synthesizing libraries of compounds onsolid-phase supports are known in the art. Solid-phase supports aretypically polymeric objects with surfaces that are functionalized tobind with subunits or monomers to form the compounds of the library.Synthesis of one library typically involves a large number ofsolid-phase supports. To make a combinatorial library, solid-phasesupports are reacted with one or more subunits of the compounds and withone or more numbers of reagents in a carefully controlled, predeterminedsequence of chemical reactions. In other words, the library subunits are“grown” on the solid-phase supports. The larger the library, the greaterthe number of reactions required, complicating the task of keeping trackof the chemical composition of the multiple species of compounds thatmake up the library. In some embodiments, the small molecules are lessthan about 2000 Daltons in size, alternatively less than about 1500,750, 500, 250 or 200 Daltons in size.

The small molecule agents described in any of the aspects herein can bederived from any type of chemical reaction that can be carried out on asolid support. Such chemical reactions include, but are not limited to,2+2 cycloadditions including trapping of butadiene; [2+3] cycloadditionsincluding synthesis of isoxazolines, furans and modified peptides;acetal formation including immobilization of diols, aldehydes andketones; aldol condensation including derivatization of aldehydes,synthesis of propanediols; benzoin condensation including derivatizationof aldehydes; cyclocondensations including benzodiazepines andhydantoins, thiazolidines, turn mimetics, porphyrins, phthalocyanines;Dieckmann cyclization including cyclization of diesters; Diels-Alderreaction including derivatization of acrylic acid; Electrophilicaddition including addition of alcohols to alkenes; Grignard reactionincluding derivatization of aldehydes; Heck reaction including synthesisof disubstituted alkenes; Henry reaction including synthesis of nitrileoxides in situ (see 2+3 cycloaddition); catalytic hydrogenationincluding synthesis of pheromones and peptides (hydrogenation ofalkenes); Michael reaction including synthesis of sulfanyl ketones,bicyclo[2.2.2]octanes; Mitsunobu reaction including synthesis of arylethers, peptidyl phosphonates and thioethers; nucleophilic aromaticsubstitutions including synthesis of quinolones; oxidation includingsynthesis of aldehydes and ketones; Pausen-Khand cycloaddition includingcyclization of norbornadiene with pentynol; photochemical cyclizationincluding synthesis of helicenes; reactions with organo-metalliccompounds including derivatization of aldehydes and acyl chlorides;reduction with complex hydrides and tin compounds including reduction ofcarbonyl, carboxylic acids, esters and nitro groups; Soai reactionincluding reduction of carboxyl groups; Stille reactions includingsynthesis of biphenyl derivatives; Stork reaction including synthesis ofsubstituted cyclohexanones; reductive amination including synthesis ofquinolones; Suzuki reaction including synthesis of phenylacetic acidderivatives; and Wittig-Horner reactions including reactions ofaldehydes, pheromones, and sulfanyl ketones.

References disclosing the synthesis of chemical libraries as well as thedeconvolution of the individual compounds of those libraries ontoindividual solid phase supports, can be found in U.S. Patent ApplicationNo. 2009/0032592; Needels et al., (1993), Proc. Natl. Acad. Sci. USA 90:10700-10704; and PCT Application Publication No. WO 97/15390, thedisclosures of which are incorporated by reference herein.

4. Inhibitory Nucleic Acids

In one aspect of this invention, the candidate complement modulatorycompound is one or more oligonucleotides targeted to a component (suchas an mRNA) of the complement system. The inhibitory nucleic acid canbe, without limitation, any of an antisense oligonucleotide, a smallinhibitory RNA (siRNA), or a ribozyme.

The oligonucleotide of the invention may be an mRNA encoding a proteincomponent of the complement system. The oligonucleotides will bind tothe mRNAs and interfere with their functions, either by mediating theirdestruction or by preventing translation into proteins. Absolutecomplementarity, although preferred, is not required. An oligonucleotidesequence “complementary” to a portion of an RNA, as referred to herein,means a sequence having sufficient complementarity to be able tohybridize with the RNA, forming a stable duplex. The ability tohybridize will depend on both the degree of complementarity and thelength of the oligonucleotide. Generally, the longer the hybridizingnucleic acid, the more base mismatches with an RNA it may contain andstill form a stable duplex. Those skilled in the art can ascertain atolerable degree of mismatch by use of standard procedures to determinethe melting point of the hybridized complex.

In general, complementary oligonucleotides manufactured to hybridizewith mRNAs for proteins of the complement system are targeted to anyregion of the mRNA, including to the 5′ untranslated region of the mRNA,to the complement of the AUG start codon, or to the 3′ untranslatedregion.

The oligonucleotides can have alternate internucleoside linkages, suchas, but not limited to, phosphorothioate (Mag at al., Nucleic Acids Res.19:1437-1441, 1991; and U.S. Pat. No. 5,644,048), peptide nucleic acidor PNA (Egholm, Nature, 3685:566-568, 1993; and U.S. Pat. No.6,656,687), phosphoramide (Beaucage, Methods Mol. Biol. 20:33-61, 1993),phosphorodithioate (Capaldi et al., Nucleic Acids Res., 28:E40, 2000).Other oligonucleotide analogs include such as, but not limited to,morpholino (Summerton, Biochim. Biophys. Acta, 1489:141-158, 1999),locked oligonucleotides (Wahlestedt et al., Proc. Natl. Acad. Sci. USA,97:5633-5638, 2000), peptidic nucleic adds or PNA (Nielsen et al., 1993;Hyrup and Nielsen, 1996) or 2-o-(2-methoxy)ethyl modified 5′ and 3′ endoligonucleotides (McKay et al., J. Biol. Chem., 274:1715-1722, 1999).All of these references are hereby expressly incorporated by reference.The nucleic acids may contain any combination of deoxyribo- andribo-nucleotides, and any combination of bases, including uracil,adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine,isocytosine, isoguanine, etc.

The complementary oligonucleotides according to the invention maycomprise at least one modified base moiety which is selected from thegroup including but not limited to 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine. N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N2-carboxypropyl)uracil, (acp3)_(w),and 2,6-diaminopurine

The complementary oligonucleotides may also comprise at least onemodified sugar moiety selected from the group including but not limitedto arabinose, 2-fluoroarabinose, xylulose, and hexose.

The complementary oligonucleotides should be at least ten nucleotides inlength, and may range from 10 to about 50 nucleotides in length, such as11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, or 50 nucleotides in length.

Assessing Efficacy of Candidate Compounds

Therapeutic efficacy of candidate complement modulatory compounds,appropriate dosages, and methods of the present invention in a givennon-human animal (such as a rodent, e.g., a mouse or a rat), can bedetermined in accordance with in vitro complement assays well known tothose having ordinary skill in the art. The complement pathway generatesnumerous specific products during the normal course of activation andsensitive and specific assays have been developed and are availablecommercially for most of these activation products, including the smallactivation fragments C3a, C4a, and C5a and the large activationfragments Bb and sC5b-9. Most of these assays utilize monoclonalantibodies that react with new antigens exposed on the complementfragment, but not on the native proteins from which they are formed,making these assays very simple and specific. ELISA technology isparticularly common, although radioimmunoassay (RIA) is also used fordetection of C3a and C5a. These latter assays measure both theunprocessed fragments and their processed fragments (i.e., the majorforms found in the circulation). Measurement of C3a provides asensitive, pathway-independent indicator of complement activation.Detection of the fluid-phase product of membrane attack pathwayactivation, sC5b-9, provides evidence that complement is being activatedto completion. Further, the classical pathway also generates C4a,measurement of which provides information about the activity of C3binhibitor and whether such an inhibitor is activating the classicalpathway.

In addition, several in vivo models of complement activation are alsoavailable to the skilled artisan for assessing a candidate compound'sability to modulate the complement system. For example, the complementsystem is implicated in ischemia-reperfusion injury which is sustainedafter an ischemic event and subsequent restoration of blood flow.Examples of injuries that can cause ischemia-reperfusion injury include,without limitation, myocardial infarction, cerebral ischemic events,intestinal ischemia, and many aspects of vascular surgery, cardiacsurgery, trauma, and transplantation. Activation of the complementsystem plays a role in the inflammatory events of ischemia-reperfusioninjury. The ischemia injury results in alterations of the cell membrane,affecting lipids, carbohydrates, or proteins of the external surfacesuch that these exposed epitopes are altered and can act as neo-antigens(modified self antigens). The involvement of the classical pathway ofcomplement to ischemia-reperfusion injury is evidenced by micegenetically deficient in either C3 or C4 that display equal protectionfrom local injury in a hindlimb and animal model of injury (Austen etal. (2003) Int J Immunopath Pharm 16:1). Additionally, complementactivation has also been shown be involved in a kidney model of ischemiainjury (Guo et al. (2005) Ann Rev Immunol 23:821).

The invention can be further understood by reference to the followingexamples, which are provided by way of illustration and are not meant tobe limiting.

EXAMPLES Example 1 Replacement of the Endogenous Mouse C5 Gene with aHuman C5 Gene

The 97 kb human C5 gene containing coding exons 2 through 42 of thehuman C5 gene replaced 75.8 kb of the murine C5 gene locus spanningcoding exons 2 through 41 and including a portion of the 3′ untranslatedregion. See FIG. 1.

A targeting construct for replacing the mouse with the human C5 gene ina single targeting step was constructed using VelociGene® geneticengineering technology (see Valenzuela et al. (2003) High-throughputengineering of the mouse genome coupled with high-resolution expressionanalysis, Nature Biotech, 21(6):652-659). Mouse and human C5 DNA wereobtained from bacterial artificial chromosome (BAC) clones bMQ-2277L16and CTD-2559119, respectively. Briefly, a linearized targeting constructgenerated by gap repair cloning containing mouse C5 upstream anddownstream homology arms flanking a 97 kb human C5 sequence extendingfrom intron 1 just upstream of coding exon 2 through coding exon 41 andthe 3′ untranslated region (genomic coordinates: GRCh38:chr9:120,952,335-121,050,276 (-strand)) and a floxed neo selectioncassette, was electroporated into F1H4 mouse embryonic stem (ES) cells(C57BL/6×129 F1 hybrid). Correctly targeted ES cells (MAID 7140) werefurther electroporated with a transient Cre-expressing vector to removethe drug selection cassette. Targeted ES cell clones without drugcassette (MAID 7141) were introduced into an 8-cell stage SW mouseembryo by the VelociMouse® method (see, U.S. Pat. Nos. 7,294,754,7,576,259, 7,659,442, and Poueymirou et al. (2007) F0 generation micethat are essentially fully derived from the donor gene-targeted ES cellsallowing immediate phenotypic analyses Nature Biotech. 25(1):91-99).VelociMice® (F0 mice fully derived from the donor ES cell) bearing thehumanized C5 gene were identified by genotyping for loss of mouse alleleand gain of human allele using a modification of allele assay (see,Valenzuela et al. (2003)).

Correctly targeted ES cell clones were identified by aloss-of-native-allele (LONA) assay (Valenzuela et al. 2003) in which thenumber of copies of the native, unmodified C5 gene were determined bytwo TaqMan™ quantitative polymerase chain reactions (qPCRs) specific forsequences in the mouse C5 gene that were targeted for deletion. The qPCRassays comprised the following primer-probe sets (written 5′ to 3′):upstream forward primer, CCTCCGGTTA ACTGGTTTGT GAT (SEQ ID NO:1);upstream reverse primer, GCAGTGAATG GTAGACTTCC CA (SEQ ID NO:2);upstream probe, FAM-AGTGACTTTA CTTTGGTTGT TCTGCTCACA-BHQ (SEQ ID NO:3);downstream forward primer, CCGGGAAAGG AAACCAAGAC (SEQ ID NO:4);downstream reverse primer, CAGCACAGAC TGAGGATCCA A (SEQ ID NO:5);downstream probe, FAM-AGACTGTCAT TCTGGCCCGG ACCT-BHQ (SEQ ID NO:6); inwhich FAM refers to the 5-carboxyfluorescein fluorescent probe and BHQrefers to the fluorescence quencher of the black hole quencher type(Biosearch Technologies). DNA purified from ES cell clones that havetaken up the targeting vector and incorporated in their genomes wascombined with TaqMan™ Gene Expression Master Mix (Life Technologies)according to the manufacturer's suggestions in a 384-well PCR plate(MicroAmp™ Optical 384-Well Reaction Plate, Life Technologies) andcycled in an Applied Biosystems Prism 7900HT, which collectsfluorescence data during the course of the PCRs and determines athreshold cycle (Ct), the fractional PCR cycle at which the accumulatedfluorescence reaches a pre-set threshold. The upstream and downstreamC5-specific qPCRs and two qPCRs for non-targeted reference genes wererun for each DNA sample. The differences in the Ct values (ΔCt) betweeneach C5-specific qPCR and each reference gene qPCR were calculated, andthen the difference between each ΔCt and the median ΔCt for all samplesassayed was calculated to obtain ΔΔCt values for each sample. The copynumber of the C5 gene in each sample was calculated from the followingformula: copy number=2×2^(ΔΔCt). A correctly targeted clone, having lostone of its native copies, will have a C5 gene copy number equal to one.Confirmation that the human C5 gene sequence replaced the deleted mouseC5 gene sequence in the humanized allele was confirmed by a TaqMan™ qPCRassay that comprises the following primer-probe sets (written 5′ to 3′):human upstream forward primer, TGCTCTTAAA GGACCATCAT CAC (SEQ ID NO:7);human upstream reverse primer, GTAAACGGAT AACGGAAAGG ATAGA (SEQ IDNO:8); human upstream probe, FAM-CCATTTCCAA ATGCACTTCC CAGC-BHQ (SEQ IDNO:9); human downstream forward primer, CCCACTGAAC TATCACAGGA AACA (SEQID NO:10); human downstream reverse primer, GGAGATGCAT TCCAGTCTCT GTTA(SEQ ID NO:11); human downstream probe, FAM-TGAAGATGAC CTCCGGATGTAACGG-BHQ (SEQ ID NO:12).

The same LONA assay is used to assay DNA purified from tail biopsies formice derived from the targeted ES cells to determine their C5 genotypesand confirm that the humanized C5 allele has transmitted through thegerm-line. Two pups heterozygous for the replacement are bred togenerate a mouse that is homozygous for the replacement of theendogenous mouse C5 gene by the human C5 gene. Pups that are homozygousfor the replacement are used for phenotyping.

The upstream junction of the murine locus and the sequence containingthe human C5 gene is designed to be within 5′-TGAAAAACCT CCATTACTACTTCTACAGAT GCCCACAGTG GTCTTGCATT CTATCCTTGT/(GCGATCGC)/TCTGCCATCTCCTACTAGGC ATGTGGGGAA GGGGAATTCA GATGATGGTT GGAAATCTGG AAATTCTTTCCTCTCTTTTG TAATTTGCCT (SEQ ID NO:13), wherein the final mouse C5nucleotide prior to the first nucleotide of the human C5 gene is thelast T in CTTGT, and the first nucleotide of the human C5 sequence isthe first T in TCTGC, which span the AsiSI site (within parentheses),wherein the precise junctions are indicated by forward slashes. Thedownstream junction of the sequence containing the human C5 gene and thefloxed neo selection cassette is designed to be within 5′-GCAAATAGGGCAGGCCACAG TGGCCTAATT AACCCACAAT GCAGCTGTCA AATATCAAAG AAGGTTTTGTGAATTAGCCT/CTCGAGATAA CTTCGTATAA TGTATGCTAT ACGAAGTTAT ATGCATGGCCTCCGCGCCGG GTTTTGGCGC CTCCCGCGGG (SEQ ID NO:14), wherein the finalnucleotide of the human C5 sequence is the T in AGCCT, and the firstnucleotide of the floxed neo selection cassette is the first C in CTCGA,the precise junction being indicated by a forward slash. The downstreamjunction of the sequence of the murine C5 locus is designed to be within5′-ATGTCTGGAA TAACTTCGTA TAATGTATGC TATACGAAGT TATGCTAGTA ACTATAACGGTCCTAAGGTA GCGAGCTAGC/CAGGCTATTA GGCTTCTGTC CCCAACTGTG GTGGCAAATAGGCCAGACCA CAGTCACCAG ATGAAGCCAT AAATGCAGCT (SEQ ID NO:15), wherein thefinal nucleotide of the floxed neo selection cassette is the second C inCTAGC, and the first nucleotide of the murine C5 locus is the first C inCAGGC, the precise junction being indicated by a forward slash. Thedownstream junction of the sequence containing the human C5 gene and themurine locus is designed to be within 5′-GCAAATAGGG CAGGCCACAGTGGCCTAATT AACCCACAAT GCAGCTGTCA AATATCAAAG AAGGTTTTGTGAATTAGCCT/CTCGAG(ATAA CTTCGTATAA TGTATGCTAT ACGAAGTTAT) GCTAGTAACTATAACGGTCC TAAGGTAGCG AGCTAGC/CA GGCTATTAGG CTTCTGTCCC CAACTGTGGTGGCAAATAGG CCAGACCACA GTCACCAGAT GAAGCCATAA ATGCAGCTGA (SEQ ID NO:16),wherein the final nucleotide of the human C5 sequence is with the “T” inAGCCT, and the first nucleotide of the mouse C5 sequence is the first“C” in CAGGC; which span a loxP site (within parentheses) that remainsafter removal of the floxed neo selection cassette, wherein the precisejunctions are indicated by forward slashes.

Example 2 Replacement of the Endogenous Mouse C3 Gene with a Human C3Gene Version 1—Replacement with Human C3 Promoter and Coding Exons 1Through 41

The human C3 gene containing 5′ regulatory elements and all of thecoding exons 1 through 41 of the human C3 gene replaced the murine C5gene locus spanning 5′ regulatory elements and all of the coding exons 2through 41. The methods described above in Example 1 were basically usedto replace the mouse C3 gene sequences with human C3 gene sequences. SeeFIG. 2A.

Preliminarily, a targeted deletion of 25 kb of the mouse C3 gene wasgenerated in mouse ES cells by replacement of coding exons 2 through 41and including 900 bp 3′ to the polyadenylation site with a floxed neocassette. The resultant mouse ES cells, 12132 ES cells, are aheterozygous C3 knockout. 12132 cells were used to generate C3 knockoutmice according to procedures known in the art.

A targeting construct was generated containing mouse C3 upstream anddownstream homology arms flanking a human C3 sequence extending from 5′regulatory elements upstream of coding exon 1 through coding exon 41 andthe 3′ untranslated region and a floxed hygro selection cassette, andelectroporated into 12132 ES cells. Correctly targeted ES cells (MAID6148) were further electroporated with a transient Cre-expressing vectorto remove the drug selection cassette. Targeted ES cell clones withoutdrug cassette (MAID 6149) were introduced into an 8-cell stage mouseembryo. F0 mice fully derived from the donor ES cell bearing thehumanized C3 gene were identified by genotyping for loss of mouse alleleand gain of human allele as described in Example 1.

Confirmation that the floxed neo cassette replaced the deleted mouse C3gene sequence in the C3 knockout 12132 ES cells was confirmed by aTaqMan™ qPCR assay that comprises the following primer-probe sets(written 5′ to 3′): 12132TU, mouse C3 intron 1: forward primer,GGCCTGATTA CATGGACCTG TC (SEQ ID NO:17); reverse primer, CCCAGGCTTGGCTGGAATG (SEQ ID NO:18); probe, FAM-TGTCCACTCT GGAAGCCCAG GC-BHQ (SEQID NO:19); 12132TD, 3′ of mouse C3 exon 41: forward primer, GCCAGGAGAGGAAGCTGGAG (SEQ ID NO:20); reverse primer, TGGCTCAGCA GTAAAGAACA C (SEQID NO:21); probe, FAM-ACAGATTGCT GTGAGCTGCC CAAA-BHQ (SEQ ID NO:22); neocassette: forward primer, GGTGGAGAGG CTATTCGGC (SEQ ID NO:23); reverseprimer, GAACACGGCG GCATCAG (SEQ ID NO:24); probe, FAM-TGGGCACAACAGACAATCGG CTG-BHQ (SEQ ID NO:25).

Confirmation that the human C3 gene sequence replaced the deleted mouseC3 gene sequence in the humanized allele was confirmed by a TaqMan™ qPCRassay that comprises the following primer-probe sets (written 5′ to 3′):mC3-1, mouse C3 promoter: forward primer, GCCAGCCTAG CCTACTTCA (SEQ IDNO:26); reverse primer, GCCACCCATC CCAGTTCT (SEQ ID NO:27); probe,FAM-CAGCCCAGGC CCTTTAGATT GCA-BHQ (SEQ ID NO:28); mC3-2, mouse C3 3′untranslated region, forward primer, TACGGTGTTA GGTTCACTAT TGGT (SEQ IDNO:29); reverse primer, GTCGCCAGCA GTCTCATACA G (SEQ ID NO:30); probe,CAL Orange-AGTGGGCATC CCTTGCCAGG C-BHQ(SEQ ID NO:31); hygro cassette:forward primer, TGCGGCCGAT CTTAGCC (SEQ ID NO:32); reverse primer,TTGACCGATT CCTTGCGG (SEQ ID NO:33); probe, FAM-ACGAGCGGGT TCGGCCCATTC-BHQ (SEQ ID NO:34); hC3-1, human C3 promoter: forward primer,GGGCCTCCTA AGTTTGTTGA GTATC (SEQ ID NO:35); reverse primer, CAGGGCTGGTTCCCTAGAAA TC (SEQ ID NO:36); probe, FAM-TACAATAGCA GGCACAGCAC CCA-BHQ(SEQ ID NO:37); hC3-2, human C3 intron 1: forward primer, GGCTGAGAGTGGGAGTCATG (SEQ ID NO:38); reverse primer, GCACTTGCCA ATGCCATTAT C (SEQID NO:39); probe, FAM-CTGCTGTCCT GCCCATGTGG TTG-BHQ (SEQ ID NO:40);hC3-3, human C3 exon 41: forward primer, CGAATGCCAA GACGAAGAGA AC (SEQID NO:41); reverse primer, GGGCACCCAA AGACAACCAT (SEQ ID NO:42); probe,CAL Orange-CAGAAACAAT GCCAGGACCT CGGC-BHQ (SEQ ID NO:43).

The same LONA assay is used to assay DNA purified from tail biopsies formice derived from the targeted ES cells to determine their C3 genotypesand confirm that the humanized C3 allele had transmitted through thegerm-line. Two pups heterozygous for the replacement are bred togenerate a mouse that is homozygous for the replacement of theendogenous mouse C3 gene by the human C3 gene. Pups that are homozygousfor the replacement are used for phenotyping.

The sequences of the junction of the murine C3 locus and the sequencecontaining the human C3 gene, the junction of the sequence containingthe human C3 gene and the floxed hygro selection cassette, and thejunction of the sequence of the floxed hygro selection cassette and themurine C3 locus are determined as described above in Example 1.

Version 2—Replacement with Human C3 Coding Exons 2 Through 41

The human C3 gene containing coding exons 2 through 41 of the human C3gene replaced the murine C5 gene locus spanning coding exons 2 through41. The methods described above in Example 1 were basically used toreplace the mouse C3 gene sequences with human C3 gene sequences. SeeFIG. 2B.

Preliminarily, a targeted deletion of 25 kb of the mouse C3 gene wasgenerated in mouse ES cells by replacement of coding exons 2 through 41and including 900 bp 3′ to the polyadenylation site with a floxed neocassette. The resultant mouse ES cells, 12132 ES cells, are aheterozygous C3 knockout.

A targeting construct was generated containing mouse C3 upstream anddownstream homology arms flanking a human C3 sequence extending fromupstream of coding exon 2 through coding exon 41 and the 3′ untranslatedregion and a floxed hygro selection cassette, and electroporated into12132 ES cells. Correctly targeted ES cells (MAID 6155) were furtherelectroporated with a transient Cre-expressing vector to remove the drugselection cassette. Targeted ES cell clones without drug cassette (MAID6156) were introduced into an 8-cell stage mouse embryo. F0 mice fullyderived from the donor ES cell bearing the humanized C3 gene wereidentified by genotyping for loss of mouse allele and gain of humanallele as described in Example 1.

Confirmation that the floxed neo cassette replaced the deleted mouse C3gene sequence in the C3 knockout 12132 ES cells, and that the human C3gene sequence replaced the deleted mouse C3 gene sequence in thehumanized allele, was confirmed by a TaqMan™ qPCR assay that comprisesthe same primer-probe sets (written 5′ to 3′) as described above inVersion 1.

The same LONA assay is used to assay DNA purified from tail biopsies formice derived from the targeted ES cells to determine their C3 genotypesand confirm that the humanized C3 allele had transmitted through thegerm-line. Two pups heterozygous for the replacement are bred togenerate a mouse that is homozygous for the replacement of theendogenous mouse C3 gene by the human C3 gene. Pups that are homozygousfor the replacement are used for phenotyping.

The sequences of the junction of the murine C3 locus and the sequencecontaining the human C3 gene, the junction of the sequence containingthe human C3 gene and the floxed hygro selection cassette, and thejunction of the sequence of the floxed hygro selection cassette and themurine C3 locus are determined as described above in Example 1.

Example 3 Complement Hemolytic Activity Assay in Mouse Serum

Complement has been implicated in ocular inflammatory and retinaldegenerative diseases (see Mullins et al. (2000) Drusen associated withaging and age-related macular degeneration contain proteins common toextracellular deposits associated with atherosclerosis, elastosis,amyloidosis, and dense deposit disease, FASEB J, 14(7):835-846).Blocking C5 cleavage by monoclonal antibody (mAb) is a possibletherapeutic strategy for these disorders (see Copland et al. (2009)Systemic and local anti-C5 therapy reduces the disease severity inexperimental autoimmune uveoretinitis, Clinical and ExperimentalImmunology, 159:303-314). For evaluation of therapies directed atcomplement, such as C5 and/or C3 antagonists, assays are necessary toevaluate the function of candidate therapeutics in vitro and in vivo inappropriate models of human diseases, disorders and conditionsassociated with complement activation.

Methods

Complement hemolytic activity was assessed using Classical ComplementAssay CH₅₀ (Gilcas et al. (2001) Classical pathway evaluation, CurrentProtocols in Immunology, Unit 13.1). Mouse blood was collected from theheart and clotted in eppendorf tubes on ice for 1 hour; serum wasseparated by centrifugation and stored at −70° C. Sheep erythrocytes (E)were sensitized by incubation with rabbit anti-sheep hemolysin for 20min at 37° C. Sensitized cells (E_(A)) were incubated for 1 hour at 37°C. with doubling dilutions of treated or control serum from mouse orhuman. Intact E_(A) cells were pelleted and supernatant was removed tomeasure absorbance at 541 nm, an index of hemolysis. E_(A) cellsincubated with buffer only (0% lysis) or with ddH₂O (100% lysis) servedas negative and positive controls respectively. Percentage of hemolysisand CH₅₀ in each well were calculated relative to the ddH₂O control(Holt et al. (2001) Targeted deletion of the CD59 gene causesspontaneous intravascular hemolysis and hemoglobinuria, Blood,98(2):442-449). Mice, including C5^(hu/hu), C5^(−/−), C3^(hu/hu),C3^(−/−), and respective control strains, were generated withVelocigene® technology as described in Examples 1 and 2. Normal,C5-depleted, and C3-depleted human sera were obtained from Quidel Corp.,and used as controls or for comparison. Complement components were addedto test if the hemolysis function can be restored. Tested sera wereincubated for 40 min at 4° C. with complement components; human C5(Sigma-Aldrich or Quidel Corp., 50 μg/ml), human C3 (Sigma-Aldrich orQuidel Corp., 10, 400, 1200 μg/ml) and murine C5 (RegeneronPharmaceuticals, 50 μg/ml). Anti-human C5 mAb (100 μg/ml) and anti-mouseC5 mAb (Hycult Biotech, 10 μg/ml) were used to confirm the specificityand utility of this system for future drug discovery.

Results

Wild-type mouse sera have approximately 10 times lower complementhemolytic activity compared to normal human sera. As shown in FIG. 3,the complement hemolytic activity, as determined using the CH₅₀ value,was at least 10 times lower in three lots of normal or wild-type mousesera as compared to two lots of human sera.

Anti-C5 monoclonal antibodies block hemolysis in a species-specificmanner. As shown in FIG. 4, an anti-mouse C5 monoclonal antibody(msC5Ab) blocked complement hemolytic activity in normal mouse serum,and an anti-human C5 mAb (C5Ab) blocked hemolytic activity in normalhuman serum and human C5-depleted serum supplemented with human C5protein, but not in normal mouse serum.

Human C5 and C3 proteins display species-specific hemolytic activity. Asshown in FIG. 5A, human C5 and C3 proteins were able to reconstitutecomplement hemolytic activity of human C5- and C3-depleted human serum,respectively.

As shown in FIG. 5B, sera from C5^(−/−) and C3^(−/−) mice showed a lackof complement activity, and humanized C5^(hu/hu) mouse serum displayedno hemolytic activity, essentially equivalent to serum from C5^(−/−) andC3^(−/−) mice. The addition of mouse C5 protein to humanized C5^(hu/hu)mouse serum rescued complement hemolytic activity, but addition of humanC5 protein to humanized C5^(hu/hu) mouse serum did not rescue complementactivity.

As shown in FIG. 5C, the lack of complement hemolytic activity ofhumanized C5^(hu/hu) mouse serum appears not to be due to a lack ofhuman C5 protein, as the amount of human C5 protein present in the serumof these mice was about one third the amount of C5 protein present innormal human serum. FIG. 5D shows the variation between male and femalehumanized C5 mice with respect to pre-dose complement C5 levels. Therange of human C5 protein present in the serum of these mice ranged from20 to 100 μg/ml, which was sufficient to show hemolytic activity inserum. Human C5 normally added in these experiments was 50 μg/ml.

Both human C5 and C3 proteins are required to reconstitute hemolyticactivity in mouse C5 knockout sera. As shown in FIG. 6, complementhemolytic activity could not be recovered by adding human C5 protein toC5^(−/−) mouse serum, whereas the addition of human C3 and C5 proteinstogether rescued hemolysis in C5^(−/−) mouse serum.

Addition of human C3 protein rescues hemolytic activity in C5^(hu/hu)mouse serum. As shown in FIG. 7, human C3 protein is sufficient toreconstitute complement hemolytic activity in serum from C5^(hu/hu)mice.

Based on the above results, humanized C5^(hu/hu) and/or C3^(hu/hu) miceare appropriate non-human animals to evaluate the PK and PD ofhuman-specific C5 and/or C3 antagonists. Based on the above results,doubly humanized C5^(hu/hu) and C3^(hu/hu) mouse serum may displaycomplement hemolytic activity without a requirement for supplementationby human C5 and/or C3 proteins, thereby constituting an appropriatemouse strain for in vivo testing of the therapeutic efficacy ofhuman-specific C5 and/or C3 antagonists.

Conclusions

Wild-type mouse serum has dramatically lower complement hemolyticactivity compared to human serum. Genetically modified mice expressinghuman C5 are functional murine C5 knockouts, possibly because mouseconvertase cannot cleave human C5 protein. Humanized C5 mouse serumneeds the addition of human C3 protein to achieve wild-type levels ofcomplement hemolytic activity.

Example 4 Uses for Humanized C5 and/or C3 Mice

Humanized C5 and/or C3 mice are useful to evaluate the pharmacodynamics(PD) of human-specific C5 and/or C3 compounds (such as, antagonists,e.g., neutralizing anti-C5 or anti-C3 antibodies).

Pharmacokinetics (PK) and PD assays in humanized C5 and/or C3 mice areperformed according to standard procedures known in the art, forexample, as described in U.S. Pat. No. 6,355,245 for an anti-C5antibody.

Humanized C5 and/or C3 mice are useful to test the in vivo therapeuticefficacy of human-specific C5 or C3 antagonists, e.g., neutralizinganti-C5 or anti-C3 antibodies, in a variety of disease models, forexample and not for limitation: dry age-related macular degeneration(AMD); wet AMD; experimental autoimmune uveoretinitis (EAU); ocularhypertension; and optic nerve injury. (See, e.g., Makrides (1998) andMollnes et al. (2006) for a list of diseases, disorders and conditionsassociated with complement activation).

Dry AMD can be induced in humanized C5 and/or C3 mice by high fat dietand blue light damage (see, e.g., Exp Eye Res. 2002, 75(5):543-553),cigarette smoke (see, e.g., Invest Ophthalmol Vis Sci. 2006,47(2):729-737), carboxyethylpyrrole (CEP) (see, e.g., Invest.Ophthalmol. Vis. Sci. 2010, 51:1276-1281; Nat Med. 2012, 18(5):791-798).

The humanized C5 and/or C3 mice can also be bred to other transgenic AMDmodels, such as, for example, ApoE (−/−) (see, e.g., Invest. Ophthalmol.Vis. Sci. 2000, 41:2035-2042), Mdm1 (see, e.g., Hum. Mol. Genet. 2008,17:3929-3941), Sod1 (−/−) (see, e.g., PNAS 2006; 103:11282-11287), LDLR(−/−) (see, e.g., Br J Ophthalmol 2005, 89:1627-1630), and DICER1 (−/−)(see, e.g., Nature. 2011; 471(7338):325-330).

Wet AMD can be induced in humanized C5 and/or C3 mice by laser-inducedchoroidal neovascularization (CNV) (see Nat Protoc. 2013,8(11):2197-2211).

EAU can be induced in humanized C5 and/or C3 mice (see, e.g., Clin ExpImmunol. 2010, 159(3): 303-314).

Ocular hypertension can be induced in humanized C5 and/or C3 mice (see,e.g., Experimental Eye Research 2006, 83:620-628).

Optic nerve injury can be induced in humanized C5 and/or C3 mice (see,e.g., J Neurotrauma 2003, 20(9):895-904.

Humanized C5 mice can be used to evaluate human-specific C5 antagonistsin a variety of disease models, for example and not for limitation:paroxysmal nocturnal hematuria; neuromyletic optica; renaltransplantation; and kidney injury. (See, e.g., U.S. Pat. No.6,355,245.)

Therapeutic efficacy assays using humanized C5 and/or C3 mice areperformed according to standard procedures known in the art, forexample, as described in U.S. Pat. No. 6,355,245 for an anti-C5antibody.

Example 5 Pharmacokinetic/Pharmacodynamic Study of an Anti-C5 Antibodyin Humanized C5 Homozygous Mice

This example utilizes male and female humanized C5 homozygous miceprepared according to the prior Examples to test the ability of ananti-C5 antibody to decrease complement activation.

Methods

A total of 25 C5^(hu/hu) mice (males and females) were used in thestudy. Serum (20 μL) was collected from two mice at each designatedtimepoint, which included a “predose” timepoint as well as timepoints at3 h, 6 h, 1 d, 2 d, 4 d, 1 W, 2 W, 4 W following subcutaneousadministration of 15 mg/kg of an anti-human C5 monoclonal antibody.Samples were stored at −20° C. until assayed for pharmacokinetics (PK)and pharmacodynamics (PD) alteration of complement activation.

Complement hemolytic activity was assessed as described above in Example3. As above, human C3 (Sigma-Aldrich or Quidel Corp., 400 and 800 μg/ml)was added to serum to recapitulate the human complement system.

Results

As shown in FIG. 8, subcutaneous injection of 15 mg/kg of an anti-humanC5 antibody decreases complement activation for at least one week postinjection. This effect was observed irrespective of whether 400 μg/ml(FIG. 8A) or 800 μg/ml (FIG. 8B) human C3 protein was added to thehemolytic assays.

As shown in FIG. 9, PK analysis of C5 monoclonal antibody revealedclearance over time of this antibody with 2 out of 5 mice havingundetectable levels by day 28.

Example 6 Cross Activity of Human and Mouse Complement Proteins

This Example investigated the cross-activity of human and mouse C3 andC5 proteins using the engineered mice described in the previousexamples. As discussed in Example 3 and as shown in FIG. 6, the human C5and the mouse C3 proteins are not sufficient to reconstitute hemolyticactivity in mice lacking endogenous C5. In this study, the ability ofmouse C5 and human C3 to restore hemolytic activity in C3 knockout micewas observed.

Methods

Complement hemolytic activity was assessed as described in Example 3.

Mice, including C5^(−/−), C3^(−/−), and respective control strains, weregenerated with Velocigene® technology as described in Examples 1 and 2.Normal, C3-depleted human sera were obtained from Quidel Corp. and usedas controls or for comparison. Complement components were added to testif the hemolysis function can be restored. Tested sera were incubatedfor 40 min at 4° C. with complement components; human C3 (Sigma-Aldrichor Quidel Corp., 50, 100, 500, 1200 μg/ml); and human C5 (Sigma-Aldrichor Quidel Corp., 50 μg/ml). Anti-human C5 mAb (100 μg/ml) and anti-mouseC5 mAb (Hycult Biotech, 10 μg/ml) were used to confirm the specificityand utility of this system for future drug discovery.

Results

As shown in FIG. 10A, addition of human C3 protein to wild type miceserum can enhance hemolytic function over that observed in wild typecontrols. However, when compared to the addition of human C3 proteinback into C3-depleted human serum, the observed enhancement is less thanthat shown in humans. Further, as illustrated in FIG. 10B, percentagehemoloysis is restored using humanized C5^(hu/hu) mouse serumsupplemented with human C3 protein.

When this experiment was repeated using sera derived from C3 knockoutanimals, it was shown that adding human C3 protein to C3^(−/−) serumrescues hemolytic function, particularly with increasing concentrationsof added human C3 (FIG. 11).

In contrast, FIG. 12 shows that addition of human C3 to C5 nullanimal-derived sera does not rescue hemolytic function.

CONCLUSIONS

In summary, the data derived from the present example demonstrates thatwhile human C3 is able to cross-react with murine C5, mouse C3 issimilarly unable to cross-react with human C5 to recapitulate complementactivity in vitro. A summary of these results is presented below inTable 1.

TABLE 1 Summary of mouse and human C3, C5 cross-activity Classicalpathway mouse +human complement activity WT C3 >WT WT C5 WT C3, C5 C3KOC3 =WT C3KO C5 C3KO C3, C5 =WT C5KO C3 0 C5KO C5 0 C5KO C3, C5 ≥WT

The invention claimed is:
 1. A rodent, comprising a replacement of arodent gene sequence comprising an exon of a rodent C5 gene at anendogenous rodent C5 locus with a nucleic acid sequence comprising exon2 through exon 41 of a human C5 gene to form a modified C5 gene, whereinexpression of the modified C5 gene is under control of rodent regulatoryelements at the endogenous rodent C5 locus, and wherein the rodent is amouse or a rat.
 2. The rodent of claim 1, wherein the nucleic acidsequence comprises all 41 exons of the human C5 gene.
 3. The rodent ofclaim 1, wherein the rodent is a mouse that is incapable of expressing amouse C5 protein.
 4. The rodent of claim 1, wherein the rodent is amouse that expresses a mouse C3 protein encoded by an endogenous mouseC3 gene.
 5. The rodent of claim 1, wherein the rodent is a mouse thatexpresses a human or humanized C3 protein.
 6. The rodent of claim 5,wherein the mouse comprises a replacement at an endogenous mouse C3locus of a mouse gene encoding a mouse C3 protein with a human C3 geneencoding a human C3 protein.
 7. The rodent of claim 1, wherein therodent is a mouse that expresses human C5 protein in serum at aconcentration of at least about 10% or 200% of the level of mouse C5protein present in the serum of an age-matched mouse that expressesfunctional endogenous mouse C5 protein, but does not comprise saidreplacement.
 8. The rodent of claim 1, wherein the rodent is a mousethat expresses human C5 protein in serum at a concentration of betweenabout 10% and about 200% of the level of mouse C5 protein present in theserum of an age-matched mouse that expresses functional endogenous C5protein, but does not comprise said replacement.
 9. The rodent of claim1, wherein the rodent is a mouse that expresses human C5 protein inserum at a concentration of between about 10 μg/ml and about 150 μg/m.10. The rodent of claim 1, wherein the rodent is a mouse that expresseshuman C5 protein in serum at a concentration of at least about 10 μg/ml.11. The rodent of claim 1, wherein the rodent is a mouse and the mousegene sequence being replaced comprises exon 2 through exon 42 of themouse C5 gene.
 12. The rodent of claim 1, wherein the rodent is a mouse,and a portion of the endogenous mouse C5 locus, including 5′ regulatoryelements upstream of exon 1 through exon 41, is deleted and replacedwith a human C5 gene sequence comprising 5′ regulatory elements upstreamof exon 1 through exon 41 of the human C5 gene.
 13. The rodent of claim1, further comprising replacement of the 3′ untranslated region of therodent C5 gene with the 3′ untranslated region of the human C5 gene. 14.The rodent of claim 12, further comprising replacement of the 3′untranslated region of the rodent C5 gene with the 3′ untranslatedregion of the human C5 gene.
 15. A method for making a humanized rodent,comprising replacing a rodent C5 gene sequence at an endogenous rodentC5 locus with a human C5 gene sequence comprising exon 2 through exon 41of a human C5 gene to form a modified C5 gene, wherein the modified C5gene is operably linked to rodent regulatory elements or sequences atthe endogenous rodent C5 locus, and wherein the rodent is a mouse or arat.
 16. The method of claim 15, wherein the human C5 gene sequencereplacing the rodent C5 gene sequence comprises all 41 exons of thehuman C5 gene sequence.
 17. The method of claim 15, wherein the rodentis a mouse and the mouse C5 gene sequence being replaced comprises exon2 through exon 42 of the mouse C5 gene.
 18. The rodent of claim 1,wherein the rodent is a mouse.
 19. The rodent of claim 1, wherein therodent is a rat.
 20. The rodent of claim 11, wherein the rodent is amouse.
 21. The rodent of claim 11, wherein the rodent is a rat.
 22. Anisolated rodent cell or tissue, whose genome comprises a replacement ofa rodent gene sequence comprising an exon of a rodent C5 gene at anendogenous rodent C5 locus with a nucleic acid sequence comprising exon2 through exon 41 of a human C5 gene to form a modified C5 gene, whereinexpression of the modified C5 gene is under control of rodent regulatoryelements at the endogenous rodent C5 locus, and wherein the rodent cellor tissue is a mouse cell or tissue, or a rat cell or tissue.
 23. Theisolated rodent cell or tissue of claim 19, wherein the rodent cell is arodent embryonic stem cell.
 24. The isolated rodent cell or tissue ofclaim 23, wherein the rodent cell is a mouse embryonic stem cell.
 25. Arodent embryo, comprising the rodent embryonic stem cell of claim 23.26. A mouse embryo, comprising the mouse embryonic stem cell of claim24.